Low oxygen affinity mutant hemoglobins

ABSTRACT

Non-naturally occurring mutant hemoglobins rHb (betaN108Q) and rHb (betaL105W) are provided that have a lower oxygen affinity than that of native hemoglobin, but high cooperativity in oxygen binding. rHb (betaN108Q) also exhibits enhanced stability against autoxidation. The mutant hemoglobins are preferably produced by recombinant DNA techniques. Such mutant hemoglobins may be used as a component of a blood substitute and hemoglobin therapeutics.

ACKNOWLEDGMENT

The present invention was developed in part with government supportunder grant numbers HL-24525 and HL-58249. The government has certainrights in this invention.

FIELD OF THE INVENTION

This invention relates generally to novel mutant hemoglobins and moreparticularly relates to recombinant mutant hemoglobins “rHb (βN108Q)”(alternative designation “rHb (β108Asn→Gln)”) and “rHb (βL105W)”(alternative designation “rHb (β105Leu→Trp”) that possess low oxygenaffinity, and high cooperativity in oxygen binding. In particular, rHb(βN108Q) exhibits increased resistance to autoxidation as compared toother known low oxygen affinity mutants. This invention further relatesto the preparation of mutant hemoglobins using recombinant DNAtechnology that are useful as substitutes for red blood cells and forhemoglobin-based therapeutics.

BACKGROUND OF THE INVENTION

The prevalence of infectious agents such as HIV and hepatitis in redblood cells of human blood products coupled with blood shortages fromlack of suitable donors has led to great interest in the development ofred blood cell substitutes, particularly human hemoglobin (“Hb”) and itsderivatives. Hemoglobin-based oxygen carriers are potential sources ofblood substitutes during emergency medical situation See for exampleWinslow, R. M., et al. Hemoglobin-Based Red Cell Substitutes, JohnsHopkins University Press, Baltimore (1992) (hereinafter “Winslow, et al.(1992)”), the disclosure of which is incorporated herein by reference.

Hemoglobin is the oxygen-carrying component of blood, circulated throughthe blood stream inside erythrocytes (red blood cells). Human normaladult hemoglobin (“Hb A”) is a tetrameric protein with a molecularweight of about 64,500 containing two identical a chains having 141amino acid residues each and two identical β chains having 146 aminoacid residues each, with each also bearing prosthetic groups known ashemes. The erythrocytes help maintain hemoglobin in its reduced,functional form. The heme-iron atom is susceptible to oxidation, but maybe reduced again by one of two systems within the erythrocyte, thecytochrome b₅, and glutathione reduction systems. For a review onhemoglobin, see, Dickerson, R. E., et al. Hemoglobin: Structure,Function, Evolution, and Pathology pp. 22-24. Benjamin/Cummings, MenloPark, Calif. (1983) (hereinafter “Dickerson, et al. (1983)”), thedisclosure of which is incorporated herein by reference.

The oxygenation process of Hb A is cooperative, i.e., the binding of thefirst oxygen molecule enhances the binding of the second, third, andfourth oxygen molecules. The oxygenation process is also regulated byinteractions between individual amino acid residues and various solutes,known as heterotropic allosteric effectors. These effectors include ionsor molecules such as hydrogen ion, chloride, carbon dioxide, inorganicphosphate, and organic polyanions, such as 2,3-bisphosphoglycerate(“2,3-BPG”) and inositol hexaphosphate (“IHP”).

Hemoglobin is able to alter its oxygen affinity, thereby increasing theefficiency of oxygen transport in the body, due to its dependence on theallosteric effector 2,3-BPG. 2,3-BPG is present within erythrocytes at aconcentration that allows hemoglobin to release bound oxygen to tissues.In the absence of 2,3-BPG, hemoglobin binds oxygen very tightly and doesnot readily release its bound oxygen. The Hb A molecule alone, were itto be introduced into a subject, would not be able to properly allowoxygen to be delivered to tissues in the body due to a lack of 2,3-BPG,which lowers the oxygen affinity of Hb, in the blood plasma. See,Winslow, et al. (1992). Any Hbs designed to be functional as Hb-basedoxygen carriers or hemoglobin therapeutics should be able to deliveroxygen efficiently, i.e., they should load and unload cooperatively asHb A does inside red blood cells.

The use of cell-free solutions of hemoglobin as a potentialoxygen-carrying red cell substitute has been investigated for a longtime. See, for example, Mulder, A. G., et al., J. Cell Comp. Physiol.5:383 (1934), the disclosure of which is incorporated herein byreference. However, the use of unmodified cell-free human hemoglobinpurified from red blood cells suffers from several limitations inaddition to contamination and supply limitations noted above, namely, anincrease in oxygen affinity due to loss of allosteric effectors, such as2,3-BPG, and dissociation of Hb tetramers into αβ dimers which arecleared by renal filtration and which can cause long-term kidney damage.See, for example, Bunn, H. F., et al. J. Exp. Med. 129:909 (1969), thedisclosure of which is incorporated herein by reference.

Human globins and hemoglobins have been expressed in the following:transgenic mice, see, for example. Chada, K., et al., Nature (London)314:377 (1985) and Townes, T. M., et al. EMBO J. 4:1715 (1985),transgenic swine as described by Swanson, M. E., et al. Bio/Technology10:557 (1992), insect cell cultures as reported by Groebe, D. R., etal., Protein Expression and Purification 3:134 (1992), yeast asdescribed by Wagenbach, M., et al. Bio/Technology 9:57 (1991) andDeLiano, J. J., et al. Proc. Natl. Acad. Sci. USA 90:918 (1993), andEscherichia coli (“E. coli”) as described by Hoffman, S. J., et al.Proc. Natl. Acad. Sci. USA 87:8521 (1990), Hernan, R. A., et al.Biochemistry 31:8619 (1992), and Shen, T. J., et al. Proc. Natl. Acad.Sci. USA 90:8108 (1993) it (hereinafter “Shen, et al. (1993)”), all thedisclosures of which are incorporated herein by reference. In manyrespects, the E. coli system is the best choice for such purposesbecause of its high expression efficiency and the ease of performingsite-directed mutagenesis.

The natural N-terminal valine residues of Hb A are known to playimportant roles in regulating oxygen affinity, the Bohr effect, andinteractions with allosteric effectors and anions as reported by Bunn,H. F., et al. eds. Hemoglobin: Molecular, Genetic and Clinical Aspects(W. B. Saunders, Co., Philadelphia, Pa.) pp. 37-60 (1986) (hereinafter“Bunn, et al. (1986)”), the disclosure of which is incorporated hereinby reference. The extra methionine can alter the N-terminal conformationof the Hb molecule as reported by Kavanaugh, J. S., et al. Biochemistry31:8640 (1992), the disclosure of which is incorporated herein byreference. Hence, the oxygenation properties of Hb depend on theintegrity of the N-terminal residue thereby mandating the removal of theextra methionine residues from the N-termini of both the α- andβ-globins of the expressed Hb before the E. coli system can be usedeffectively for the production of desired unmodified and mutant Hbs.

The cooperative oxygenation of Hb, as measured by the Hill coefficient(“n_(max)”) is a convenient measure of its oxygenation properties. SeeDickerson, et al. (1983). Hb A has an n_(max) value of approximately 3in its binding with O₂ under usual experimental conditions. Humanabnormal Hbs with amino acid substitutions in the α₁β₂ (or α₂β₁) subunitinterface generally result in high oxygen affinity and reducedcooperativity in O₂ binding compared to Hb A. See, for example,Dickerson, et al. (1983); Bunn, et al (1986) and Perutz, M. F., et al.Mechanisms of Cooperativity and Allosteric Regulation in Proteins pp.19-29 Cambridge University Press (1990), the disclosure of which isincorporated herein by reference.

Hb A in its oxy form (Hb A with oxygen molecules) has a characteristichydrogen bond between α94Asp and β102Asn in the α₁β₂ subunit interfaceas reported by Shaanan, B., et al. T. Mol. Biol. 171:31 (1983), thedisclosure of which is incorporated herein by reference (hereinafter“Shaanan, et al. (1983)”). Human Hbs with an amino acid substitution ateither the α94Asp position such as Hb Titusville (α94Asp→Asn)(Schneider, R. G., et al. Biochim. Biophys. Acta. 400:365 (1975), thedisclosure of which is incorporated herein by reference) or the β102Asnposition such as Hb Kansas (β102Asn→Thr) (Bonaventura, J., et al. T.Biol. Chem. 243:980 (1968), the disclosure of which is incorporatedherein by reference), as well as others with mutations in the α₁β₂subunit interface, exhibit very low oxygen affinity. However, all theseHb mutants which directly disrupt the hydrogen bond between α94Asp andβ102Asn in the oxy form of Hb show greatly reduced cooperativity in thebinding of oxygen and additionally dissociate easily into dimers when inthe ligated state.

It has also been shown that during the transition from the deoxy-to theoxy-state, the α₁β₂ subunit of Hb A undergoes a sliding movement, whilethe α₁β₁ subunit interface remains nearly unchanged (e, Perutz, M. F.Nature 228:726 (1970) (“Perutz (1970)”); Baldwin, J. M., et al. T. Mol.Biol. 129:175 (1979); Baldwin, J. M., J. Mol.

Biol. 136:103 (1980); Shaanan, et al. (1983); and Fermi, G., et al. J.Mol. Biol. 175:159 (1984), (“Fermi, et al., (1984)”), the disclosures ofwhich are incorporated herein by reference. There are specific hydrogenbonds, salt bridges, and non-covalent interactions that characterizeboth subunit interfaces. The Hb molecule also has a lower oxygenaffinity in the deoxy quaternary structure (T-structure) than in the oxyquaternary structure (R-structure) See, Dickerson, et al. (1983).

Low oxygen affinity human mutant Hbs which do not involve either α94Aspor , β102Asn also exist. For example, Hb Presbyterian (β108Asn→Lys)(Moo-Penn, W. F., et al. FEBS Lett. 92:53 (1978) and O'Donnell, J. K.,et al. J. Biol. Chem. 269:27692 (1994) (hereinafter “O'Donnell, et al.(1994)”); Hb Yoshizuka (β108Asn→Asp) O'Donnell, et al. (1994) andrecombinant Hb Mequon (β41Phe→Tyr) (Baudin, V., et al. Biochim. Biophys.Acta. 1159:223 (1992), the disclosures of which are incorporated hereinby reference, all exhibit low oxygen affinity compared to Hb A, but theyall exhibit a variable amount of cooperativity as measured by the Hillcoefficient, with n varying from 1.8 to 2.9. Tsai, C.-H., et al.Biochemistry 38:8751 (1999) (hereinafter, “Tsai et al. (1999)”) reportHb (α96Val→Trp, β108Asn→Lys) which has low oxygen affinity and a greatertendency to switch to the T quaternary structure. Jeong, S. T., et al.,Biochemistry 38:13433 (1999) (hereinafter, “Jeong, et al. (1999)”)report that Hb (α29Leu→Phe, α96Val→Trp, β108Asn→Lys) exhibits low oxygenaffinity and high cooperativity combined with resistance toautoxidation.

Shen, et al. (1993) and U.S. Pat. No. 5,753,465, the disclosures ofwhich are incorporated herein by reference, describe an E. coliexpression plasmid (pHE2) in which synthetic human α- and β-globin genesare coexpressed with the E. coli methionine aminopeptidase gene underthe control of separate tac promotors. E. coli cells transformed withthis plasmid express recombinant Hb A (hereinafter “rHb A”) from whichthe N-terminal methionines have been effectively cleaved by thecoexpressed methionine aminopeptidase. The resulting rHb A which lacksan N-terminal methionine is identical to the native Hb A in a number ofstructural and functional properties.

Kim, H.-W., et al. Proc. Natl. Acad. Sci. USA 91:11547 (1994)(hereinafter “Kim, et al. (1994)”), and U.S. Pat. No. 5,843,888, thedisclosures of which are incorporated herein by reference, describe anon-naturally occurring mutant hemoglobin (rHb (α96Val→Trp) (alternativedesignation “rHb (αV96W)”) that has a lower oxygen affinity than that ofnative hemoglobin, but high cooperativity in oxygen binding.

There remains a need, however, for additional mutant hemoglobin speciesthat can be used as a component of a hemoglobin-based blood substituteor therapeutic agent. Of particular interest is a mutant hemoglobin thatpossesses low oxygen affinity, high cooperativity in oxygen binding, andincreased stability against autoxidation. There is a further need forsuch a hemoglobin produced by recombinant methods and an efficientexpression system for producing such a mutant hemoglobin in high yield,especially for use in a blood substitute product or hemoglobintherapeutics.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the present invention to providemutant human hemoglobins with low oxygen affinity and high cooperativityin oxygen binding.

Another object of the present invention is to provide mutant hemoglobinswith low oxygen affinity, high cooperativity in oxygen binding, andincreased stability against autoxidation.

Another object of the present invention is to provide non-naturallyoccurring mutant human hemoglobins with low oxygen affinity and highcooperativity in oxygen binding.

Another object of the present invention is to provide non-naturallyoccurring mutant human hemoglobins with low oxygen affinity, highcooperativity in oxygen binding, and increased stability againstautoxidation.

Another object of the present invention is to provide non-naturallyoccurring mutant human hemoglobins with low oxygen affinity, highcooperativity in oxygen binding, and preferably with stability againstautoxidation that are produced artificially, preferably by recombinantmeans, and that have the correct heme conformation.

Another object of the present invention is to provide mutant hemoglobinsthat in a cell-free environment have similar oxygen binding propertiesas those of human normal adult hemoglobin in red blood cells.

Yet another object of the present invention is to provide mutanthemoglobins with low oxygen affinity and high cooperativity in oxygenbinding in which the T-structure is stabilized while the R-structure isundisturbed.

Still another object of the present invention is to provide artificialhemoglobins for use as a hemoglobin-based oxygen carrier/red bloodsubstitute or therapeutic agent.

These and other objects of the present invention are achieved by one ormore of the following embodiments.

In one aspect, the invention features a non-naturally occurring mutanthuman hemoglobin wherein the asparagine residue at position 108 of theβ-chains is replaced by a glutamine residue.

In a preferred embodiment, the hemoglobin possesses low oxygen affinityas compared to human normal adult hemoglobin, high cooperativity inoxygen binding, increased stability against autoxidation, and isproduced recombinantly.

In another aspect, the invention features an artificial mutanthemoglobin which in a cell-free enviornment has oxygen bindingproperties comparable to those of human normal adult hemoglobin in redblood cells wherein said hemoglobin contains a mutation such that theasparagine residue at position 108 of the β-chains is glutamine.

A non-naturally occurring low oxygen affinity mutant hemoglobin withincreased stability against autoxidation that has oxygen bindingproperties comparable 5 to those of human normal adult hemoglobin in thepresence of the allosteric effector 2,3-bisphosphoglycerate, wherein theasparagine residue at position 108 of each of the β-chains is replacedby a glutamine residue.

In yet another aspect, the invention features a non-naturally occurringmutant human hemoglobin wherein the leucine residue at position 105 ofthe β-chains is replaced by a tryptophan residue.

In a preferred embodiment, the hemoglobin possesses low oxygen affinityas compared to human normal adult hemoglobin, high cooperativity inoxygen binding, and is produced recombinantly.

In another aspect, the invention features an artificial mutanthemoglobin which in a cell-free environment has oxygen bindingproperties comparable to those of human normal adult hemoglobin in redblood cells wherein said hemoglobin contains a mutation such that theleucine residue at position 105 of the β-chains is tryptophan.

A non-naturally occurring low oxygen affinity mutant hemoglobin that hasoxygen binding properties comparable to those of human normal adulthemoglobin in the presence of the allosteric effector2,3-bisphosphoglycerate, wherein the leucin residue at position 105 ofeach of the β-chains is replaced by a tryptophan residue.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiment, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a cDNA sequence (SEQ ID NO: 5) for the alpha- and beta-globingenes for rHb (βN108Q) derived from plasmid pHE7009.

FIG. 1B is a cDNA sequence (SEQ ID NO: 7) for alpha- and beta-globingenes for rHb (βL105W) derived from plasmid pHE7004.

FIGS. 2A and 2B show the FPLC profiles of rHb (βN108Q) (peak b) (FIG.2A) and rHb (βL105W) (peak b) (FIG. 2B).

FIGS. 3A and 3B show the pH dependence of the oxygen affinity (P₅₀) andthe Hill coefficient (n_(max)), respectively, of rHb (αL29F) (□); rHb(βN108Q) (Δ); rHb (αL29F, βN108Q) (X); rHb (βL105W) (▾); and Hb A (O) in0.1 M sodium phosphate buffer at 29° C. Oxygen dissociation data wereobtained with 0.1 mM Hb.

FIG. 4 shows oxygen-binding curves of rHb (βN108Q); rHb (αL29F, βN108Q);rHb (βL105W); and Hb A with and without the presence of allostericeffector, 5 mM 2,3-BPG, in 0.1 M phosphate buffer at pH 7.4 and 29° C.Protein concentration was 0.1 mM heme.

FIG. 5 shows the autoxidation of Hb A (O); rHb (βN108Q) (▾); rHb(βL105W) (Δ); rHb (αV96W) (∇); rHb (αV96W, βN108K) (▴); rHb (αL29F,βN108Q) (⋄); and rHb (αL29F, αV96W, βN108K) (□) in PlasmaLyte buffer inthe presence of 5 mM EDTA and 5% D₂O at pH 7.4 and 37° C., Theautoxidation process was measured by monitoring the rate ofdisappearance of the oxy-marker at −2.34 ppm upfield from DSS by 300-MHz¹H-NMR.

FIGS. 6A and 6B are 500-MHz ¹H-NMR spectra showing exchangeable protonresonances (FIG. 6A) and ring-current shifted proton resonances (FIG.6B), respectively, of Hb A; rHb (βN108Q), and rHb (αL29W, βL108Q), allin the CO form in 0.1 M sodium phosphate buffer at pH 7.0 and 29° C.

FIGS. 7A and 7B are 300-MHz ¹H-NMR spectra showing ferroushyperfine-shifted N_(δ)H resonances of proximinal histidines andhyperfine-shifted and exchangeable proton resonances, respectively, ofrHb A; rHb (αL29F); rHb (βN108Q); and rHb (αL29W, βN108Q), all in the COform, in 0.1 M sodium phosphate buffer at pH 7.0 and 29° C.

FIGS. 8A and 8B are 500-MHz spectra showing the exchangeable protonresonances of rHb (βN108Q) in the CO form in 0.1 M sodium phosphatebuffer at pH 7.0 at 500 MHz at various temperatures (7° C., 11° C., 17°C., 23° C., 29° C.), without an allosteric effector (FIG. 8A) and with 4mM inositol hexaphosphate (“IHP”) (FIG. 8B).

FIGS. 9A and 9B are 500-MHz spectra showing the exchangeable protonresonances of rHb (αL29F, βN108Q) in the CO form in 0.1 M sodiumphosphate buff at pH 7.0 at various temperatures (7° C., 11° C., 17° C.,23° C., 29° C.), without (FIG. 9A) and with (FIG. 9B) 4 mM IHP.

FIGS. 10A and 10B are 600-MHz ¹H-NMR spectra showing exchangeable protonresonances (FIG. 10A) and ringcurrent shifted proton resonances (FIG.10B) of 3-6% solutions of Hb A; rHb (βL05W); rHb (αD94A, βL105W); andrHb (αD94A) in the CO form in 0.1 M sodium phosphate at pH 7.0 and 29°C.

FIGS. 11A and 11B are 600-MHz 2D heteronuclear multiple-quantumcoherence (“HMQC”) spectra of 5-8% solutions of ¹⁵N-labeled rHb (βL105W)(FIG. 11A) and Hb A (FIG. 11B) in the CO form in 90% H₂O/10% D₂O in 0.1M sodium phosphate at pH 7.0 and 29° C.

FIGS. 12A-12D are 600-MHz 2D NOESY-HMOC (“NOESY”-nuclear Overhauserenhancement spectroscopy) spectra of a 5% solution of ¹⁵N-labeled rHb(βL105W) in the CO form in 90% H₂O/10% D₂O in 0.1 M sodium phosphate atpH 7.0 and 29° C. recorded at various mixing times: 15 ms (FIG. 12A); 30ms (FIG. 12B); 60 ms (FIG. 12C); and 100 ms (FIG. 12D).

FIGS. 13A-13C are 300-MHz ¹H-NMR spectra of 3-6% solutions of Hb A; rHb(βL105W); rHb (αD94A, βL105W); and rHb (rHb (αD94A) in the deoxy form in0.1 M sodium phosphate at pH 7.0 and 29° C. FIG. 13A showshyperfine-shifted N_(δ)H resonances of proximal histidines acquired at300-MHz; FIG. 13B shows hyperfine shifted and exchangeable protonresonances acquired at 300-MHz; and FIG. 13C shows exchangeable protonresonances acquired at 300-MHz. Since rHb (αD94A, βL105W) and rHb(αD94A) easily form met-Hb during the oxygenization process, a smallamount of sodium dithionite was added to these NMR samples to diminishthe formation of met-Hb.

FIG. 14 is a 600-MHz 2D HMQC spectrum of 5-8% solutions of ¹⁵N-labeledrHb (βL105W) in the deoxy form in 90% H₂O/10% D₂O in 0.1 M sodiumphosphate at pH 7.0 and 29° C.

FIGS. 15A-15D are 600-MHz 2D NOESY-HMQC spectra of 5% solution of¹⁵N-labeled rHb (βL105W) in the deoxy form in 90% H₂O/10% D₂O in 0.1 Msodium phosphate at pH 7.0 and 29° C. recorded at various mixing times:15 ms (FIG. 15A); 30 ms (FIG. 15B); 60 ms (FIG. 15C); and 100 ms (FIG.15D). The solid line between two cross peaks indicates the inter-residueNOE effect between the ¹H_(ε1) of one residue and the ¹H_(δ1) and¹H_(ζ2) of the other residue for β37Trp and β105Trp.

FIGS. 16A-16B show exchangeable proton resonances in 600-MHz ¹H-NMRspectra of 3-6% solutions of Hb A; rHb (βL105W); rHb (αD94A, βL105W);and rHb (αD94A) in the CO form in 0.1 M sodium phosphate at pH 7.0 andat 11°, 20°, and 29° C. in the absence (FIG. 16A) and presence (FIG.16B) of 2 mM IHP.

FIGS. 17A-17D are 600-MHz 2D heteronuclear single-quantum cohere(“HSQC”) spectra of 5-8% solutions of ¹⁵N-labeled rHb (βL105W) in the COform in 90% H₂O/10% D₂O in 0.1 M sodium phosphate at pH 7.0 and varioustemperatures in the absence and presence of 2 mM IHP. FIG. 17A—29° C. inthe absence of IHP; FIG. 17B—at 29° C. in the presence of 2 mM IHP; FIG.17C—at 20° C. in the presence of 2 mM IHP; and FIG. 19D—11° C. in thepresence of 2 mM IHP.

FIGS. 18A-18B show ring-current shifted proton resonances 600-MHz ¹H-NMRspectra of 3-6% solutions of Hb A; rHb (βL105W); rHb (αD94A, βL105W);and rHb (αD94A) in the CO form in 0.1 M sodium phosphate at pH 7.0 andvarious temperatures in the absence (FIG. 18A) and presence (FIG. 18B)of 2 mM IHP.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

As used herein, “Hb A” or “native Hb A” means human normal adulthemoglobin as obtained from human subjects. “Recombinant human normaladult hemoglobin,” “rHb A,” and “unmodified rHb A” mean human normaladult hemoglobin produced through recombinant DNA technology and havingessentially the same structure and function as native Hb A as describedby Shen, et al. (1993), and U.S. Pat. No. 5,753,465.

“rHb (βL105W)” refers to a recombinant mutant human hemoglobin in whichthe leucine residue at position 105 of each of the β-chains of the Hbmolecule has been replaced by a tryptophan residue. This hemoglobinpossesses low oxygen affinity and high cooperativity in oxygen bindingcompared to Hb A. rHb (βL105W) is designed to form a hydrogen bondbetween α94Asp and β105Trp in the α₁β₂ subunit interface in order tolower the oxygen affinity by stabilizing its deoxy quaternary structure.

“rHb (βN108Q)” refers to a mutant human hemoglobin produced throughrecombinant DNA technology in which the asparagine residue at position108 of the β-chains located in the α₁β₂ interface and in the centralcavity of the Hb molecule, have been replaced by glutamine residues.This hemoglobin possesses low oxygen affinity and high cooperativity inoxygen binding, and also increased resistance to autoxidation ascompared to other known recombinant low oxygen affinity mutanthemoglobins, such as, rHb (αV96W) and rHb (αV96W, βN108K).

“Autoxidation” refers to the turning or conversion of oxyhemoglobin(“HbO₂” or “oxy-Hb”) into methemoglobin (“met-Hb”). In HbO₂ theheme-iron atoms are in the reduced ferrous (Fe²⁺) state, however, inmet-Hb, the heme-iron atoms are in the oxidized ferric (Fe³⁺) state.

“Deoxy” and “oxy” refer to the oxygenation state of the heme-iron atomin Hb A and rHbs. Oxyhemoglobin (“oxy-Hb” or “HbO2”) has four oxygenmolecules bound to the heme groups; deoxyhemoglobin (“deoxy-Hb”)contains no oxygen molecules. In normal arterial blood, normal adulthemoglobin A (“Hb A”) is in the oxy form (“Hb O2 A” or “oxy-Hb A”). Invenous blood, a portion of Hb A is in the deoxy form (“deoxy-Hb A”).“Carbonmonoxy-Hb,” “HbCO A,” “rHbCO,” and “CO form” all refer tohemoglobin bound to carbon monoxide molecules rather than oxygenmolecules.

“Ferri-hemoglobin,” “ferri-Hb,” “ferric form,” “methemoglobin,”“met-Hb”, and “Fe⁺³ state” all refer to hemoglobin with their respectiveheme-iron atoms oxidize to the ferric (Fe³⁺) state. Ferri-Hb does notbind oxygen.

“Methionine aminopeptidase” refers to the enzyme methionineaminopeptidase which specifically cleaves the amino-(N) terminalmethionine residue from a peptide sequence.

“Oxygen affinity” means the strength of binding of oxygen to ahemoglobin molecule. High oxygen affinity means hemoglobin does notreadily release its bound oxygen molecules. The P₅₀ is a measure ofoxygen affinity.

“Cooperativity” refers to the binding of oxygen by the four subunits ofthe hemoglobin molecule and is measured by the Hill coefficient(n_(max)). For Hb A in 0.1 M sodium phosphate at pH 7.4 and 29° C.,n_(max) is about 3.2.

The two classical quaternary structures are the T (tense) quaternary Dstructure for the low affinity deoxy-Hb and the R (relax) quaternarystructure for the high affinity oxy-Hb. “R-type” or “R-like,” andsimilar terms refer to those hemoglobins which exhibit characteristicquaternary structural markers, such as the proton resonance at 10.7 ppmfrom DSS on ¹H-NMR spectra. “T-type” or “T-like” and similar terms referto those hemoglobins which exhibit characteristic T quaternarystructures, such as the t4 proton resonance at ˜14.0 ppm from DSS on¹H-NMR spectra.

II. METHODS AND RESULTS

Using the Escherichia coli expression system described by Shen, et al.(1993); U.S. Pat. No. 5,753,465; and Kim, et al. (1995); U.S. Pat. No.5,843,888, new non-naturally occurring artificial recombinanthemoglobins (“rHbs”) have been constructed, having low oxygen affinitywhile maintaining high cooperativity in oxygen binding. One of the rHbs,rHb (βN108Q) also exhibits increased resistance to autoxidation ascompared to certain other known low oxygen affinity mutants. Moreparticularly, the present invention is directed to: a recombinantlyproduced mutant of Hb A, denoted herein as rHb (βN108Q), in which theasparagine residues at position 108 of each of the β-chains (SEQ ID NO:8), located in the α₁β₁ subunit interface and in the central cavity ofthe Hb molecule, have been replaced by glutamine residue; and arecombinantly produced mutant of Hb A, denoted herein as rHb (βL105W) inwhich the leucine residues at position 105 of each of the β chains (SEQID NO: 8) have been replaced by tryptophan and in this molecule a newhydrogen bond is formed from β105Trp to β94Asp in the α₁β₂ subunitinterface in order to lower the oxygen binding affinity by stabilizingits deoxy quaternary structure.

These new artificial hemoglobins, i.e., derived entirely from sourcesother than blood, possess a low oxygen affinity and high cooperativityin oxygen binding. Additionally, rHb (βN108Q) exhibits increasedresistance to autoxidation as compared to other known low oxygenaffinity mutants, such as rHb (αV96W) and rHb (αV96W, βN108K). Further,these new artificial hemoglobins exhibit no unused subunitdisassociation when ligated. In a cell-free environment the rHbs of thepresent invention have similar or lower oxygen binding properties tothose of Hb A in red blood cells. Such rHbs therefore are of value ashemoglobin-based oxygen carriers, i.e., potential blood substitutes, orhemoglobin therapeutics.

It is also within the scope of the present invention to prepare and useother low oxygen affinity hemoglobins with other appropriate mutations.In particular, the methods of the present invention may be used toproduce other mutant hemoglobins with additional advantageousproperties. Methods for evaluating the suitability of other usefulmutants possessing the properties of such low oxygen affinity, highcooperativity, and increased resistance to autoxidation for use in ablood substitute or therapy are described herein below. The preferredmaterials and methods for obtaining rHb (βL105W) and rHb (βN108Q) aregiven in the following reference examples. While the rHbs of the presentinvention are preferably produced recombinantly, it is understood thatnon-recombinant methods may also be used.

The preferred mutant rHbs of the present invention, rHb (βL105W) and rHb(βN108Q), can switch from the R quaternary structure to the T quaternarystructure in their ligated state upon the addition of an allostericeffector, IHP, and/or by lowering the temperature. The recombinanthemoglobins of the present invention can therefore be used to gain newinsight regarding the nature of subunit interactions in the α₁β₂ andα₁β₁ interfaces and the molecular basis for the allosteric mechanism ofhemoglobin.

As will be shown below, rHb (≈N108Q) of the present invention shows alow oxygen affinity, an enhanced Bohr effect, but a similarcooperativity as that of Hb A, and also exhibits slower autoxidation tomethemoglobin (“met-Hb”) as compared to other known low oxygen affinityrecombinant hemoglobins such as, for example, rHb (α96Val→Trp) and rHb(α96Val→Trp, β108Asn→Lys) (Kim, H.-W., et al. Biochemistry35:6620-6627(1996) (hereinafter “Kim, et al. (1996)”); Ho, C., et al. BloodSubstitutes: Present and Future Perspectives of Blood Substitutes(Tsuchida, E., Ed.), Elsevier Science SA, Lausanne, Switzerland, pp.281-296 (1998) (hereinafter “Ho, et al. (1998)”); Jeong, et al. (1999);and Tsai, et al. (1999), the disclosures of which are incorporatedherein by reference), oxidize much faster. Therefore, rHb (βN108Q) canbe useful for hemoglobin-based oxygen carriers and hemoglobintherapeutics.

Proton nuclear magnetic resonance (“¹H-NMR”) studies show that rHb(βN108Q) has similar tertiary structure around the heme pockets andquaternary structure in the α₁β₁ and α₁β₂ subunit interfaces as comparedto those of Hb A. ¹H-NMR studies also demonstrate that rHb (βN108Q) canswitch from the R quaternary structure to the T quaternary structurewithout changing its ligation state upon the addition of an allostericeffector, IHP, and/or by lowering the temperature. This suggests thatthe T quaternary structure of rHb (βN108Q) is more stable than that ofHb A. This is consistent with the molecular mechanism of low-oxygenaffinity found in rHb (αV96W) (Kim, H.-W., et al., T. Mol. Biol. 248:867(1995) (hereinafter “Kin, et al. (1995)”); U.S. Pat. No. 5,843,888) andrHb (αV96W, βN108Q) (Ho, et al. (1998); Tsai, et al. (1999)).

It has been reported by Carver, T. E., et al. J. Biol. Chem. 267: 14443(1992); Brantley, R. E. Jr., et al. J. Biol. Chem. 268: 6995 (1993)(hereinafter “Brantley, et al. (1993)”; and Eich, R. F., et al.Biochemistry 35: 6976 (1996), the disclosures of which are incorporatedherein by reference, that substitution of the Leu residue forphenylalanine at the B10 position can inhibit autoxidation in myoglobinand that at the B10 position of the α-chain can lower NO reaction withdeoxy- and oxy-Hb A. Reduction of the NO reaction with oxy-Hb A byappropriate mutations, i.e., αL29F, in the distal heme pocket has beenassociated with reduction of the hypertensive effect recorded in vivo(Doherty, D. H., et al. Nature Biotech. 16: 672 (1998), the disclosureof which is incorporated herein by reference). Hence, as detailed below,such mutation was further introduced into βN108Q to produce a doublemutant, rHb (αL29F, βN108Q). It was found that this double mutant ismore stable against autoxidation as compared to rHb (βN108Q), butexhibits comparable oxygen binding properties to those of Hb A in thepresence of allosteric effector, 2 mM 2,3-BPG.

Mutant rHb (βL105W) was designed to form a new hydrogen bond fromβ105Trp to α94Asp in the α₁β₂ subunit interface in order to lower theoxygen binding affinity by stabilizing its deoxy quaternary structure.It was found that rHb (βL105W) possesses a very low oxygen affinity andmaintains high cooperativity (P₅₀=28.2 mm Hg, n_(max)=2.6 in 0.1 Msodium phosphate at pH 7.4 and 29° C.) as compared to Hb A (P₅₀=9.9 mmHg, n_(max)=3.2 in 0.1 M sodium phosphate at pH 7.4 and 29° C.). MutantrHb (αD94A, βL105W) and rHb (αD94A) were designed to provide evidencethat rHb (βL105W) forms a new hydrogen bond from β105Trp to α94Asp inthe α₁β₂ subunit interface of the deoxy quaternary structure. Themultinuclear, multidimensional nuclear magnetic resonance (“NMR”)studies performed in accordance with the present invention in¹⁵N-labeled rHb (βL105W) have identified the resonance of the indolenitrogen-attached proton of β105Trp for rHb (βL105W). ¹H-NMR studies onHb A and mutant rHbs were used to investigate the structural basis forthe low oxygen affinity rHb (βL105W). NMR results show that rHb (βL105W)forms a new hydrogen bond from β105Trp to α94Asp in the α₁β₂ subunit ofthe deoxy quaternary structure. It is believed that the low oxygenaffinity of rHb (βL105W) is due to the formation of a new hydrogen bondbetween β105Trp and α94Asp in the deoxy quaternary structure.

Proton nuclear magnetic resonance (“NMR”) spectroscopy was used to studythe tertiary and quaternary structures of Hbs in solution (Ho, et al.(1992)). Several exchangeable proton resonances at ˜15 to ˜9 ppm fromthe methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate(“DSS”) have been characterized as intersubunit H-bonds in the algi andα₁β₂ subunit interfaces in both oxy and deoxy states of Hb A. TheseH-bonded protons observed by NMR can be used as structural markers infunctional studies. In particular, the resonance at ˜14 ppm from DSS hasbeen identified as the inter-subunit H-bond between α42Tyr and β99Asp inthe α₁β₂ interface of deoxy-Hb A, a characteristic feature of theT-structure of Hb A (Fung, L. W.-M., et al. Biochemistry 14:2526 (1975)(hereinafter “Fung, et al. (1975)”), 1975; Russu, I. M., et al. Biochem.Biophys. Acta 914:40 (1987) (hereinafter “Russu, et al. (1987)”). Byobserving this T-structure marker in both the deoxy and the CO forms ofHbs under various conditions, the stability of the T-conformation can beassessed and the transition from the T- to the R-structure can bemonitored.

In the present invention, the strategy for designing rHbs with lowoxygen affinity and high cooperativity was to stabilize the T-structurewhile not perturbing the R-structure. (See Ho, et al. (1998); Tsai, etal. (1999)). This strategy has been demonstrated in the design of rHb(αV96W), which has low oxygen affinity and normal cooperativity (Kim etal. (1995); U.S. Pat. No. 5,843,888). This designed mutation is locatedat the α₁β₂ subunit interface and in the central cavity of the Hbmolecule.

According to ¹H-NMR studies, rHbCO (αV96W) can switch from theR-structure to the T-structure without changing its ligation state whenthe temperature is lowered and/or when IHP, an allosteric effector, isadded. The crystal structure of rHb (αV96W) in its T-state has shown anovel water-mediated H-bond between α96Trp Nε₁ and β101Glu Oε₂ in theα₁β₂ subunit interface (Puius, T. A., et al. Biochemistry 37: 9258(1998) (hereinafter “Puius”, et al. (1998)”). Both ¹H-NMR studies andthe crystal structure indicate that the T-structure of this rHb isstabilized. In the present invention, the NMR studies have also shownthat rHbCO (βN108Q) and rHbCO (βN105W) can switch to the T quaternarystructure even when they are still ligated. These results suggest thatthe T structure of these two rHbs are more stable than that of Hb A.

As stated above, the methods of the present invention may also be usedto produce other mutant artificial hemoglobins with different propertiesas well as hemoglobins with mutations that compensate for mutants thatare naturally occurring. The preferred materials and methods forobtaining rHb (βN108Q) and rHb (βL105W) are given in the followingreference example. Non-recombinant methods may also be used.

REFERENCE EXAMPLE Construction of Expression Plasmids for rHb (βN108Q)and rHb (βL105W)

The E. coli Hb A expression plasmids pHE2 and pHE7, which respectivelycontain human α- and β- globin genes and cDNAs, were used as thestarting plasmids for expressing the mutant hemoglobins of the presentinvention. The construction of plasmids pHE2 and pHE7 and properties ofthe rHb A produced thereby are fully described in Shen, et al. (1993),U.S. Pat. No. 5,753,465, and Shen, T.-J., et al. Protein Eng. 10: 1085(1997) (hereinafter “Shen, et al. (1997)”), Kim, et al. (1994), and U.S.Pat. No. 5,843,888), the disclosure of which is incorporated herein byreference.

The construction of plasmid pHE2009 for expression of mutant r Hb(βN108Q) using synthetic globin genes was carried out as follows. Theplasmid pHE2 was used as the starting plasmid and an oligonucleotide ofsequence 5′-CGTCTGCTGGGTCAGGTACTAGTTTGCG-3′(SEQ ID NO: 1) (mutated codonis underlined) was purchased from DNA International, Inc. (Lake Oswego,Oreg.) and used as a primer to introduce the mutation βN108Q into pHE2.Techniques for oligonucleotide synthesis are well known and thisinvention is not limited to any particular technique. The site-directedmutagenesis procedure followed the protocol of an “Altered Sites IIIn-Vitro Mutagenesis System” kit (Promega Corporation, Madison, Wis.)and the resultant plasmid pHE2009 contained the expected mutation3N108Q.

The construction of plasmids pHE2020 (mutant rHb (αD94A) and pHE2004(mutant rHb βL105W)) using synthetic globin genes was similar to that ofpHE2009, except the mutation oligonucleotide 5′-CTGCGTGTTGCTCCGGTCAACTTCAAACTG-3′(SEQ ID NO: 2, mutated codon αD94A isunderlined) and 5′GGAAAACTTCCGA TGGCTGGGTAACGTAC-31′(SEQ ID NO: 3,mutated codon βL105W is underlined) were used. Both oligonucleotideswere purchased from DNA International, Inc. (Lake Oswego, Oreg.).

The construction of plasmid pHE2017 (mutant rHb (αD94A, βL105W)) wasaccomplished by ligating the 0.51-kb SmaI-PstI fragment of pHE2020 withthe 6.34-kb kb PstI-SmaI fragment of pHE2004. The construction ofplasmid pHE2018 for expression of mutant rHb (αL29F, βN108Q) wasaccomplished by ligating the 6.06-kb PstI-BamHI fragment of pHE2009 withthe 0.79-kb BamHI-PstI fragment of pHE284. The construction of plasmidpHE284 containing the mutation αL29F from plasmid pHE2 was reportedpreviously by Jeong, et al. (1999).

The construction of plasmid pHE7009 for expression of mutant rHb(βN108Q) using the human globin cDNAs was carried out as follows. Thecoding sequences of human α- and β-globin cDNAs in plasmid pHE7 wereinserted into pTZ18U (Bio-Rad Laboratories, Hercules, Calif.) by methodswell known in the art. Site-directed mutagenesis was performed asdescribed by Kunkel, T. M. et al., Proc. Natl. Acad. Sci. USA 82:488(1985) the disclosures of which are incorporated herein by reference,and Shen, et al. (1993). An oligonucleotide of sequence 5′-ACAGACCAGTACTTGTCC CAGGAGCCT-3′ (SEQ ID NO: 4) (mutated codon Asn→Gln isunderlined) was purchased from DNA International, Inc. (Lake Oswego,Oreg.), and used as the mutation primer. The human normal β-globin cDNAin plasmid pHE7 was then replaced with the mutated cDNA to produceplasmid pHE7009. The DNA sequences for the α- and β-globin cDNAs inpHE7009 are shown in FIG. 1A (SEQ ID NO: 5). The amino acid sequence forthe human beta chains of hemoglobin is shown in SEQ ID NO: 8. PlasmidpHE7009 in host cell E. coli JM109 and designated pHE7009/JM109 wasdeposited with the American Type Culture Collection of Manassas, Va. onApr. 27, 2000 under number PTA-1768.

The construction of plasmid pHE7004 for expression of mutant rHb(βL105W) using the human globin cDNAs was carried out in the similar wayas that of plasmid pHE7009, except an oligonucleotide of sequence5′-CCTGAGAACTTC AGGTGGCTAGGCAACGTGCTGGTC-3′ ((SEQ ID NO: 6), mutatedcodon Leu→Trp is underlined) was purchased from DNA International, Inc.(Lake Oswego, Oreg.) and used as the mutation primer. The DNA sequencesof the α- and β-globin cDNAs in pHE7004 are shown in FIG. 1B (SEQ ID NO:7). The amino acid sequence for the human beta chains of hemoglobin isshown in SEQ ID NO: 8. Plasmid pHE7004 in host cell E. coli JM109 anddesignated pHE7004/JM109 was deposited with the American Type CultureCollection of Manassas, Va. on Apr. 27, 2000 under number PTA-1769.

Growth of Cells

Plasmids pHE7009, and pHE7004 were individually transformed in E. colistrain JM109 (Promega, Madison, Wis.) by methods well known in the art.E. coli cells were grown in Terrific Broth (“TB”) medium plus 100 μg/mLampicillin in a 10-liter Microferm fermenter (New Brunswick Scientific,Model BioFlo 3000) at 32° C. until the optical density at 600 nm reached10. TB medium contains 1.2% bactotryptone, 2.4% bactoyeast extract,0.17M KH₂PO₄, 0.072M K₂HPO₄, and 1% glucose solution. Expression of rHbswas induced by adding isopropyl β-thiogalactopyranoside (Sigma, St.Louis, Mo.) to a concentration of 0.1-0.4 mM. The culture was thensupplemented with hemin (20-50 mg/liter) (Sigma) and the growth wascontinued for at least another 4 hr. The cells were harvested bycentrifugation and stored frozen at −80° C. until needed forpurification. For details, refer to Shen, et al. (1993), and Shen, etal. (1997).

Although E. coli cells are presently preferred for expressing andproducing the recombinant mutant hemoglobin of the present invention,the invention is not limited to E. coli cells. Other appropriateexpression systems such as yeast, insect cells and transgenic animalssuch as pigs, sheep, and cows may also advantageously be used to expressmutant hemoglobins. Plasmids pHE7009 and pHE7004 have been optimized forE. coli cells, but other expression systems may be advantageously used.The plasmids can also be constructed with human genes.

Isolation and Purification of rHbs

The recombinant hemoglobins obtained from cells transformed withplasmids pHE7009 and pHE7004 were purified as essentially described byShen, et al. (1993), and Shen, et al. (1997). The frozen stored cellpaste was put into lysis buffer (40 mM trisbase/1 mM benzamidine)(Sigma) at 3 ml/gm of cell paste). The cell lysis procedure was used topass the cell paste through a high-pressure homogenizer (ModelEmulsiFlex-C5, Avestin) 3 times. The lysate was then centrifuged at 4°C. for 2.5 hours at 13,000 rpm in a Beckman centrifuge (Beckman JA14rotor). The supernatant from the lysate was saturated with CO gas andleft at 30° C. overnight as described by Tsai, et al. (1999). Thesupernatant was then put through a Millipore Minitan AcrylicUltrafiltration system to concentrate the protein. Polyethyleneimine(Sigma) was added to a final concentration of 0.5% to precipitatenucleic acids. After centrifugation, the sample was dialyzed against 20mM Tris-HCl/0.5 mM triethylenetetraamine (“TETA”) (Sigma) at pH 8.3overnight with one or two changes of buffer. Throughout the aboveprocedures, the sample was kept at 4° C. and maintained in a COatmosphere. Following the procedures of Shen, et al. (1993) and Shen, etal. (1997), the rHb fraction collected after passage through aQ-Sepharose Fast-Flow column (Pharmacia Anion Exchanger) Pharmacia wasoxidized and reduced, and converted to the CO form. This Hb solution wasthen purified by eluting through a fast protein liquid chromatography(“FPLC”) Mono-S column (Pharmacia Cation Exchanger, HR 16/10) with agradient of 10 mM sodium phosphate in 0.1-0.5 mMethylenediaminetetraacetic acid (“EDTA”) at pH 6.8 (eluent A) and 20 mMsodium phosphate in 0.1-0.5 mM EDTA at pH 8.3 (eluent B).

rHb (βN108Q) was eluted individually in two major peaks. FIG. 2A showspeak a and peak b for rHb (βN108Q). FIG. 2B shows rHb (βL105W) waseluted individually in three major peaks, peaks a, b, and c. rHbscollected from peak b in both cases contained less than 2% methionine atthe amino-termini and with the correct molecular weight.

Mass Spectrometry

Hb samples subjected to mass spectrometry were dialyzed extensivelyagainst distilled H₂O and then lyophilized. Immediately before analysis,the samples were dissolved in water to a concentration of 125 pmol of Hbper μl of H₂O (7.8 mg/ml). Aliquots of these solutions were then dilutedto give a final concentration of 10 pmol/μl of 50:50 water/ acetonitrilecontaining 0.2% formic acid. Aliquots (10 μl) of these final solutionswere introduced into the electrospray ion source at 5 μl/minute.

The electrospray ionization analyses were performed on a VG Quattro-BQ(Fisons Instruments, VG Biotech, Altrincham, U.K.), as described byShen, et al. (1993). Automated cycles of Edman degradation wereperformed on an Applied Biosystems gas/liquid-phase sequencer (Model470/900A) equipped with an on-line phenylthiohydantoin amino acidanalyzer (Model 120A). These two analytical procedures were used toassess the quality of the rHbs. All rHbs used in this study had thecorrect molecular weights and contained less than 2% of methionine atthe amino termini.

Oxygen-Binding Properties of rHbs

Oxygen dissociation curves of rHbs were measured by a Hemox-Analyzer(TCS Medical Products, Huntington Valley, Pa.) at 29° C. as a functionof pH. The concentration of Hb used was approximately 0.1 mM per heme.The methemoglobin (“met-Hb”) reductase system described by Hayashi, A.,et al. Biochem. Biophys. Acta310:309 (1973), the disclosure of which isincorporated herein by reference, was used if needed to reduce theamount of met-Hb in the sample. A visible absorption spectrum of eachsample was recorded immediately after oxygen equilibrium measurement,and the met-Hb content was estimated by using the extinctioncoefficients of Hb reported by Antonini, E., Physiol. Rev. 45:123(1965), the disclosure of which is incorporated herein by reference.Oxygen equilibrium parameters were derived by fitting Adair equations toeach equilibrium oxygen-binding curve by a nonlinear least-squaresprocedures. P₅₀, a measure of oxygen affinity, was obtained at 50%saturation. The Hill coefficient (n_(max)), a measurement ofcooperativity, was determined from the maximum slope of the Hill plot bylinear regression. n_(max) was derived between 60% and 65% oxygensaturation. The accuracy of P₅₀ measurements in mm Hg is ±5% and thatfor n_(max) is ±7%.

¹H-NMR Spectroscopy Measurements of rHbs

¹H-NMR spectra of rHbs were obtained on Bruker AVANCE DRX-300, AVANCEDRX-500, and AVANCE DRX-600 NMR spectrometers that were operated at 300,500, and 600 MHz, respectively, and at temperatures ranging from 10°C.-36° C. All of the Hb samples were placed in 0.1 M sodium phosphatebuffer (in 100% H₂O) at pH 7.0. The Hb concentration range wasapproximately 5% (˜3 mM in terms of heme). The water signal wassuppressed by using the “jump-and-return” pulse sequence as reported byPlateau, P., et al. J. Am. Chem. Soc. 104:7310 (1982) (hereinafter“Plateau, et al. (1982)”), the disclosure of which is incorporatedherein by reference. Proton chemical shifts are referenced to the methylproton resonance of the sodium salt of 2,2-dimethyl-2-silapentane-5sulfonate (“DSS”) indirectly by using the water signal, which signaloccurs at 4.76 ppm downfield from that of DSS at 29° C., as the internalreference.

Autoxidation of rHbs

The autoxidation of rHbs was recorded by monitoring the disappearingrate of the oxy-marker (−2.34 ppm from DSS) from Bruker AVANCE DRX-300¹H-NMR spectra. The autoxidation reaction was carried out in PlasmaLytebuffer (Baxter) (5% D₂O) with 5 mM EDTA at pH 7.4 and at 37° C. The HbO₂concentration was 5% (˜3 mM in terms of heme).

FUNCTIONAL STUDIES

Oxygen-binding Properties of rHbs

FIGS. 3A and 3B show the oxygen-binding measurements of rHb (αL29F), rHb(βN108Q), rHb (α29F, βN108Q), rHb (βLI05W), and Hb A in 0.1 M sodiumphosphate buffer as a function of pH at 29° C. rHb (βN108Q) exhibits asignificantly lower oxygen affinity as compared to that of Hb A over thepH range from pH 6.79 to pH 8.09. The oxygenation process of rHb(,N108Q) is very cooperative with an n_(max) value of about 2.7 to 3.1depending on the pH, compared to about 3.2 for Hb A (FIG. 3B). On theother hand, the mutation at the α-chain B10 position, i.e., αL29F,increases the oxygen affinity and decreases the cooperativity. rHb(αL29F, βN108Q) shows slightly higher P₅₀ values as compared to those ofHb A at pH <7.4, suggesting that the effect of the mutations on theoxygen affinity is additive. rHb (αL29F, βN108Q) preserves cooperativityin binding of oxygen with an n_(max) value of 2.4 to 2.8 (FIG. 3B). rHb(βL105W) exhibits very low oxygen affinity (about 2-3 times lower) andmaintains normal cooperativity from pH 7.0 to 8.0 as compared to Hb A.

FIG. 4 shows that the oxygen affinities of rHb (βN108Q) and also rHb(βL105W) measured in the absence of 2,3-BPG are lower than that of Hb Ain the presence of 5 mM 2,3-BPG, making them potential candidates for anoxygen carrier in a blood substitute system. FIGS. 3A and 3B also showthat the alkaline Bohr effect (which, in Hb A, results in a decrease inoxygen affinity with a lowering of the pH) is enhanced in rHb (βN108Q)and rHb (αL29F, βN108Q) compared to Hb A.

Table 1 below compares the number of Bohr protons released uponoxygenation per heme calculated from the linkage equation ΔH⁺=−∂logP₅₀/∂pH. (Wyman, J., Adv. Protein Chem. 4:407 (1948) and Adv. ProteinChem. 19:233 (1964), (hereinafter “Wyman, J. (1948) and (1964)”) thedisclosures of which are incorporated herein by reference. Both rHb(βN108Q) and rHb (αL29F, βNI08Q) release more Bohr protons than Hb A.

TABLE 1 Bohr effect of Hb A, rHb (βN108Q), rHb (αL29F, βN108Q), and rHb(βL105W) in 0.1 M sodium phosphate buffer at 29° C. Hemoglobin δlogP₅₀/δpH in 0.1 phosphate Hb A 0.48 (pH 6.79-8.00) rHb (βN108Q) 0.56 (pH6.79-8.09) rHb (αL29F, βN108Q) 0.67 PpH 6.79-7.97) rHb (βL105W) 0.55 (pH7.00-8.00)

Autoxidation.

The autoxidation process was monitored for oxy-Hb A, oxy-rHb (βN108Q),oxy-rHb (αL29F, βN108Q), oxy-rHb (βLI05W) and three other knownlow-oxygen affinity mutants, oxy-rHb (αV96W), oxy-rHb (αV96W, βN108K)and oxy-rHb (αL29F, αV96W, βN108K), by a 300-MHz NMR spectrometer. Theresonance at −2.34 ppm upfield from DSS has been assigned to the γ₂-CH₃of E11Val of Hb A in the oxy form of Hb A (Dalvit, C., et al.,Biochemistry 24:3398 (1985), the disclosure of which is incorporatedherein by reference). Monitoring the rate of disappearance of theoxy-marker (−2.34 ppm from DSS) as a function of time allows for thedetermination of the autoxidation rate of the Hb samples. The resultsare shown in FIG. 5. The percentage of ferrous-Hb varies with time (t)mono-exponentially and the autoxidation rate constant can be obtainedfrom: [ferrous-Hb]_(t)=[ferrous-b]_(t=0)×exp (−k_(auto)×t), wherek_(auto) is the autoxidation rate constant. The autoxidation rateconstants of Hb A and rHbs are listed in Table 2 below. At pH 7.4 and37° C. in PlasmaLyte buffer, rHb (βN108Q), rHb (βL105W), rHb (αV96W),and rHb (αV96W, βN108K) autoxidized 2.8-, 8-, 4.4-, and 8-times fasterthan Hb A. rHb (βN108Q) is shown be to more stable against autoxidationthan other known low-oxygen affinity mutants developed in thelaboratory; i.e., rHb (αV96W), rHb (βL105W), and rHb (αV96W, ON108K).The autoxidation rate is slowed down by introducing the mutation αL29Finto rHb (βN108Q) and rHb (αV96W, βN108K). rHb (αL29F, βNI08Q) and rHb(αL29F, αV96W, βN108K) autoxidized 2.5-and 2.8-times slower than rHb(βN108Q) and rHb (αV96W, βN108K), respectively. Thus, the mutation αL29Fis very effective in slowing down the autoxidation process as suggestedby the results on myoglobin (Brantley, et al., (1993), the disclosure ofwhich is incorporated herein by reference). Hemichrome-like spectra areobserved in the autoxidation process of only rHb (αL29F, αV96W, βN108K)among all the low oxygen affinity rHbs studied. Hemichrome forms whenmethemoglobin (met-Hb) converts from the ferric high-spin form to theferric low-spin form in which the distal imidazole displaces the H₂Oligand (Levy et al., Biochemistry 29: 9311 (1990); Levy, et al.,Biophys. J. 61: 750 (1992); Blumberg, et al., Adv. Chem. Series 100: 271(1991)). This is in accordance with the results from Jeong et al. (1999)in which the oxidized form of rHb (αL29F, αV96W, βN108K) exhibits ofhemichrome-like spectra, making it undesirable to be considered as acandidate for an oxygen carrier.

TABLE 2 Autoxidation rate constants, oxygen affinity and cooperativityof low-oxygen affinity mutants. Hemoglobin k_(auto) (h⁻¹)^(a) P₅₀ (mmHg)^(b) n_(maxn) ^(b) Hb A 0.0158 ± 0.0002 9.64 3.28 rHb (βN108Q) 0.0449± 0.0007 17.46 3.10 rHb (αL29F, βN108Q) 0.0181 ± 0.0006 12.06 2.77 rHb(βL105W)  0.123 ± 0.0048 28.2 2.60 rHb (αV96W) 0.0689 ± 0.0008 16.382.94 rHb (αV96W, βN108K)  0.125 ± 0.0051 50.65 2.36 rHb (αL29F, αV96W,0.0449 ± 0.0014 21.97^(c) 1.81^(c) βN108K) ^(a)Rate constants for theautoxidation (kauto) of Hb A and r Hbs are obtained at [oxy-Hb] 3 mMheme in PlasmaLyte buffer at pH 7.4 and 37° C. ^(b)Oxygen affinity andcooperativity were obtained in 0.1 M sodium phosphate buffer at 29° C.and pH 7.4. Protein concentrations were 0.1 mM heme. ^(c)From Jeong, etal. (1999).

STRUCTURAL STUDIES OF rHb (βN108Q) AND rHb (αL29F, βN108K) ¹H-NMRInvestigations

¹H-NMR spectroscopy is an excellent tool for monitoring changes in thetertiary and quaternary structures of Hb A and its variants (see e.g.,Shen, et al. (1993); Kim, et al. (1994); Kim, et al. (1995); Kim, et al.(1996); and Barrick, D., et al. Nat. Struct. Biol. 4:78 (1997), thedisclosures of which are incorporated herein by reference). FIG. 6Ashows the exchangeable proton resonances and FIG. 6B shows thering-current-shifted resonances of Hb A, rHb (βN108Q), and rHb (αL29F,βN108Q) in the CO form measured at 500 MHz. The ring-current-shiftedresonances are sensitive to the orientation and/or conformation of theheme group relative to the amino acid residues in the heme pockets,i.e., the tertiary structure of the Hb molecule (see, Ho, C., Adv.Protein Chem. 43:153 (1992), (hereinafter “Ho (1992)”), the disclosureof which is incorporated herein by reference). The resonances at ≈−1.8and ≈−1.7 ppm have been assigned to the γ₂-CH₃ of the E11Val of theβ-chain and α-chain of HbCO A, respectively (Lindstrom et al. (1972);Dalvit et al. (1985)). These two resonances are not changed in rHbCO(βN108Q). However, the resonance assigned to the γ₂-CH₃ of the α-E11Valof rHbCO (αL29F, βN108Q) is shifted upfield to ≈2.0 ppm, suggesting thatthe γ₂-CH₃ group of the α-E11 valine residues in rHbCO (αL29F, βN108Q)is located closer to the normal of the heme than in HbCO A. α29L is inclose proximity to E11Val, hence, the amino acid substitution αL29F isexpected to alter the conformation of the distal heme pocket site of theα-chain. There are some other changes in the ring-current-shiftedresonances among these rHbs. The experience has been that minordifferences in the intensity and positions of ring-current-shiftedresonances are common features in many rHb mutants. (See, for example,Shen, et al. (1993); Kim, et al. (1994); Kim, et al. (1995); and Kim, etal. (1996); Ho, et al. (1998); Sun, D. P., et al. Biochemistry 36:6663(1997) (hereinafter “Sun, et al. (1997)”), the disclosure of which isincorporated herein by reference; and Tsai, et al. (1999)). Thesechanges reflect slight adjustments of the conformation of the hemesand/or the amino acid residues in the heme pockets as the result of themutation.

The exchangeable proton resonances of the Hb molecule arise from theexchangeable protons in the subunit interfaces. Of special interest tothe present invention are the exchangeable proton resonances at 14.2,12.9, 12.1, 11.2, and 10.7 ppm from DSS, which have been characterizedas the inter-subunit H-bonds in the α₁β₁ and α₁β₂ subunit interfaces inboth deoxy (T) and/or oxy (R) states of Hb A (Russu, et al (1987); Fung,et al. (1975)); and Ho (1992), the disclosures of which are incorporatedherein by reference). The resonances at 12.9 ppm and 12.1 ppm from DSShave been assigned to the H-bonds between α122His and β35Tyr, andα103His and P131GIn, respectively (see Russu, et al. (1987) andSimplaceanu, et al. Biophys. J. (79:1146) (2000) (hereinafter“Simplaceanu, et al. (2000)”). In the spectra of rHbCO (βN108Q) andrHbCO (αL29F, βN108Q) (as seen in FIG. 6A), three resonances instead ofone occur corresponding to the chemical shift of HbCO A at 12.1 ppm. Themain peak occurs at 12.0 ppm, with a shoulder at 11.8 ppm and an extraresonance at 12.3 ppm. The intensities of the resonances at 12.3 and11.8 ppm are not even 1/10 of the ones at 12.0 ppm and at 12.9 ppm,indicating that these two extra resonances are unlikely to becontributed by additional protons. The sum of the integrated areas ofthe resonances at 11.8, 12.0, and 12.3 ppm is about the same as the areaof the single resonance at 12.9 ppm, suggesting the coexistence of threeconformers of rHb (βN108Q) in CO form.

FIG. 7A shows the hyperfine-shifted and FIG. 7B shows the exchangeableproton resonances of rHbs and Hb A in the deoxy form in 0.1 M phosphateat pH 7.0 and 29° C. The resonance at 63 ppm from DDS has been assignedto the hyperfine-shifted N_(δ)H-exchangeable proton of the proximalhistidine residue (α87His) of the α-chain of deoxy-Hb A and the one at77 ppm from DSS has been assigned to the corresponding residue of theβ-chain (β92His) of deoxy-Hb A (Takahashi, S., et al. Biochemistry19:5196 (1980) and La Mar, G. N., et al. Biochem. Biophys. Res. Commun.96:1172 (1980), the disclosures of which are incorporated herein byreference). The chemical shift positions of these two proximal histidylresonances in rHb (βN108Q) are exactly the same as those of Hb A,indicating no perturbations around the proximal histidine residues ofthis rHb. However, the resonance at 63 ppm from DSS of rHb (αL29F) andrHb (αL29F, βN108Q) is shifted 4 ppm downfield to 67 ppm, reflecting achange in the environment of the proximal heme pocket of the α-chain m)as a result of the mutation at αL29F.

The spectral region from 10-25 ppm arises from the hyperfine-shiftedresonances of the porphyrin ring and the amino acid residues situated inthe proximity of the heme pockets and the exchangeable protonresonances. There are no noticeable differences seen in the resonancesfrom 10-25 ppm between deoxy-Hb A and deoxy-rHb (βN108Q). However, thereare spectral changes in rHb (αL29F) and rHb (αL29F, βNI08Q) over theregion from 16-20 ppm, reflecting changes in the environment of the hemepockets of both the α- and the β-chains. The resonance at 14.2 ppm hasbeen identified as the inter-subunit H-bond between α42Tyr and β99Asp inthe α₁β₂ interface in deoxy-Hb A (Fung, et al. (1975)), a characteristicfeature of the deoxy (T) quaternary structure of Hb A (Perutz, (1970)).This resonance of rHb (αL29F) and rHb (αL29F, βN108Q) is shifted 0.5 ppmupfield to 13.7 ppm, indicating that this α₁β₂ interface H-bond in thedeoxy form is perturbed by the mutation at αL29F.

A unique feature of the rHbs of the present invention with low oxygenaffinity and high cooperativity is the appearance of the T-marker at14.2 ppm on lowering the temperature and/or adding IHP to these rHbs inthe CO form (see Kim, et al. (1995); Ho, et al. (1998); Tsai, et al.(1999)). Studies on the temperature dependence of exchangeable protonresonances of rHbs in the CO form can be used to assess the structuraleffect on oxygen affinity. FIGS. 8A and 8B and FIGS. 9A and 9B show theexchangeable proton resonances of rHb (βN108Q) and rHb (αL29F, βNI08Q)in the CO Go form in the absence (FIGS. 8A, 9A) and presence (FIGS. 8B,9B) of 4 mM IHP in 0.1 M sodium phosphate buffer as a function oftemperature. The resonance at 14.2 ppm of rHb (βN108Q) is observablestarting at 23° C. in 0.1 M phosphate at pH 7.0 in the presence of 4 mMIHP (FIG. 8B). The appearance of the T-marker in the presence of 4 mMIHP and at low temperature in the spectra of CO-ligated rHb (βN108Q) andrHb (αL29F, βN108Q) indicates that the T-states of rHb (βN108Q) and rHb(αL29F, βN108Q) are more stable than that of Hb A. However, thisresonance in the spectra of Hb (αL29F, βN108Q) has a much smallerintensity than that in the spectra of rHb (βN108Q) at low temperature,i.e., 11° C. and in the presence of 4 mM IHP.

STRUCTURAL STUDIES OF rHb (βL105W)

rHb (βL105W) was designed to form a new hydrogen bond with α94Asp in theα₁β₂ subunit interface in order to lower the oxygen binding affinity bystabilizing its deoxy quaternary structure. rHb (αD94A, βL105W) and rHb(αD94A) were constructed to provide evidence that β105Trp of rHb(βL105W) does form a new hydrogen bond with α94Asp in the α₁β₂ subunitinterface of the deoxy quaternary structure. Multinuclear,multidimensional nuclear magnetic resonance (NMR) studies on ¹⁵N-labeledrHb (βL105W) have identified the indole nitrogen-attached protonresonance of β105Trp for rHb (βL105W). ¹H-NMR studies were used toinvestigate the structural basis for the low oxygen affinity of rHb(βL105W).

¹H-NMR Studies of the Structures of rHbs in the CO Form

FIG. 10A shows the exchangeable proton resonances of Hb A, rHb (βL105W),rHb (αD94A, βL105W), and rHb (αD94A) in the CO form. The exchangeableproton resonances arise from the exchangeable protons in the subunitinterfaces. Recent multinuclear, multidimensional NMR studies on the¹⁵N-labeled rHb A have assigned the resonances at 10.6, 10.4 and 10.1ppm to β37Trp, α14TIP and β15Trp, respectively (Simplaceanu, et al.(2000)). The crystal structure of Hb A in the oxy form (Shaanan, (1983))suggested the likely candidate to form an H-bond with β37Trp in the α₁β₂subunit interface is α94Asp. The spectrum of rHb (βL105W) in the CO formshows an additional proton resonance in the region of exchangeableproton resonances (FIG. 10A). Since residues β037 and β105 are bothlocated in the α₁β₂ interface and are close in the R-quaternarystructure (Shaanan, (1983)), the replacement of Leu by Trp at β105position may cause the proton resonance of β37Trp to shift away from itsoriginal chemical shift. It is suspected that the extra resonance (ateither 11.0 ppm or 10.8 ppm) originates from β105Trp. Heteronuclear,two-dimensional (“2D”) NMR studies on the ¹⁵N-labeled rHb (βLI05W) were,therefore, carried out to assign these resonances in the spectrum of rHb(βL105W). The spectrum of rHb (αD94A) in the CO form shows that theresonance at 10.6 ppm (assigned to β037Trp in Hb A) is missing and a newresonance shows up at 9.7 ppm compared to the spectrum of Hb A (FIG.10A). This result suggests that the shift of the resonance of β37Trp at10.6 ppm to 9.7 ppm (closer to the water resonance) is due to the lackof an H-bond between α94 and β37 in rHb (αD94A) in the CO form. Thisresult also confirms the assignment of the resonance at 10.6 ppm to theinter-subunit H-bond between α94Asp and β037Trp. The spectrum of rHb(αD94A, βL105W) in the CO form shows that one extra proton resonanceappeared at 10.8 ppm compared to the spectrum of rHb (αD94A). Theresonance at 10.8 ppm was assigned to the indole NH of β105Trp of rHb(αD94A, βL105W) and rHb (βL105W).

FIG. 10B shows the ring-current-shifted proton resonances of Hb A, rHb(βLI05W), rHb (αD94A, βL105W), and rHb (αD94A) in the CO form. Thering-current-shifted resonances are very sensitive to the hemeconformation and the tertiary structure of the heme pockets (Ho,(1992)). The spectrum for the ring-current-shifted proton resonances ofrHb (βL105W) in the CO form differs only slightly from that of Hb A,while the spectra of rHb (αD94A, βL105W) and rHb (αD94A) are verydifferent from that of Hb A. These differences imply that someadjustments of the heme conformation and/or the amino acid residues inthe heme pockets occurred due to the mutation αD94A. Previous studieshave shown that minor differences in the ring-current-shifted resonancesare common features in many mutant rHbs. (Kim, et al. (1994); Kim, etal. (1995); Kim, et al. (1996); Sun, et al. (1997)).

Heteronuclear 2D NMR Studies on ¹⁵N-labeled rHb (βL105W) in the CO Form

In order to assign the proton resonances at 11.0 ppm and 10.8 ppm in the¹H-NMR spectrum of rHb (βL105W), heteronuclear 2D NMR studies on¹⁵N-labeled rHb (βL105W) in the CO form were performed. The results areshown in FIGS. 11A and 1B and FIGS. 12A-12D. FIGS. 11A and 11B show the600-MHz HMQC spectra of ¹⁵N-labeled rHb (βL105W) and rHb A in the COform. A doublet is observed at the (¹Hε₁, ¹⁵Nε₁) chemical shiftcoordinates for Trp residues because this spectrum was acquired without¹⁵N decoupling. In general, the ¹Hε₁ resonances of Trp residues usuallyappear at ˜9 to ˜12 ppm (Cavanagh, et al. (1996); BioMagResBank (1999)(www.bmrb.wisc.edu/ref₁₃ info/statsel.htm) in the proton dimension, andtheir ¹⁵Nε₁ resonances usually appear at ˜121 to ˜133 ppm(BioMagResBank) in the ¹⁵N dimension. The ¹⁵N chemical shifts for theproton resonances at 11.0 ppm and 10.8 ppm in the ¹H-NMR spectrum of rHb(βLI05W) are at 134 ppm and 129 ppm, respectively, suggesting that theseresonances originate from a Trp residue. Since the chemical shift of aproton is much easier to be affected than that of nitrogen by itsenvironment, the assignment of (11.0, 134) ppm to β37Trp and (10.8, 129)ppm to β105Trp was made (FIGS. 11A and 11B). This also agrees with whatis shown in FIG. 10A. The HMQC spectrum also correlates the Trp ¹⁵Nε₁chemical shifts with the carbon-bound proton ¹Hδ₁. As shown in FIGS. 11Aand 11B, the ¹Hδ₁ cross-peaks at (7.3, 129) and (7.1, 127) ppm areobserved for α14Trp and β15Trp, respectively, in the spectra of both¹⁵N-labeled rHb (βL105W) and rHb A in the CO form. Also observed are¹Hδ₁ cross-peaks (through two-bond coupling) at (7.4, 134) ppm with muchweaker intensity for β37Trp in the spectrum of ¹⁵N-labeled rHb A in theCO form (results not shown in FIG. 11B). Since the ¹Hδ₁ cross-peaks forβ37Trp and β105Trp cannot be seen in the spectrum of ¹⁵N-labeled r Hb(βLI05W) in the CO form (FIG. 11A), NOESY-HMQC experiments have beenperformed at different mixing times to provide more evidence for thepresent Trp assignments. As shown in FIGS. 12A-12D, the ¹Hδ₁ and ¹Hζ₂cross-peaks of all four Trp residues can be seen even at short mixingtimes. The intensities of these cross-peaks become weaker when themixing time was 60 or 100 ms (FIGS. 12C, 12D). FIGS. 12A-12D also showthat the chemical shifts of ¹Hδ₁ and ¹Hζ₂ cross-peaks are very close toeach other for β37rrp and β105Trp. All these results confirm theassignments for the Trp residues.

¹H-NMR Studies of the Structures of rHbs in the Deoxy Form

FIG. 13A shows the hyperfine-shifted N_(δ)H resonances of proximalhistidines in the 300-MHz ¹H-NMR spectra of Hb A, rHb (βL105W), rHb(αD94A, βL105W), and rHb (αD94A) in the deoxy form. The spectrum for thehyperfine-shifted N_(δ)H resonances of proximal histidines of mutantrHbs in the deoxy form is very similar to that of Hb A. FIG. 13B showsthe hyperfine-shifted and exchangeable proton resonances in the 300-MHz¹H-NMR spectra of Hb A, rHb (βL105W), rHb (αD94A, βL105W), and rHb(αD94A) in the deoxy form. The hyperfine-shifted proton resonances arisefrom the protons on the heme groups and their nearby amino acid residuesdue to the hyperfine, interactions between these protons and unpairedelectrons of Fe(II) in the heme pocket (Ho (1992). The hyperfine-shiftedproton resonances of rHb (βL105W) in the region +24 to +16 ppm are verysimilar to those of Hb A, but those for rHb (αD94A, βL105W) and rHb(αD94A) are somewhat different from these for Hb A. FIG. 13C shows theexchangeable proton resonances in the 300-MHz ¹H-NMR spectra of Hb A,rHb (βL105W), rHb (αD94A, βL105W), and rHb (αD94A) in the deoxy form.The ¹H resonance at ˜14 ppm has been identified as the intersubunitH-bond between α42Tyr and β99Asp in the α₁β₂ interface in deoxy-Hb A, acharacteristic feature of the deoxy (T)-quaternary structure of Hb A(Fung, et al. (1975)). The resonance at ˜12.2 ppm has been assigned tothe H-bond between α3His and β131 Asp at the α₁β₂ interface (unpublishedresults). The resonance at ˜11.1 ppm has been tentatively assigned tothe H-bond between α94Asp and β37Trp at the α₁β₂ interface (Fung, et al.(1975); Ishimori, et al. (1992)). Recent heteronuclear, multidimensionalNMR studies on the ¹⁵N-labeled rHb A have assigned the resonance at˜13.0 ppm to α122His, and confirmed the assignment of the resonance at˜11.1 ppm to β37Trp (unpublished results). The spectrum of rHb (βL105W)in the deoxy form shows an additional proton resonance appearing in theregion of the exchangeable proton resonances (FIG. 13C). This suggeststhat the extra resonance at 12.7 ppm originates from β105Trp. Due to thelack of an H-bond between residues α94 and β37 in rHb (αD94A) in thedeoxy form, the resonance for β37Trp should shift away from its originalchemical shift and closer to the water resonance (similar to what wasobserved in its CO form). The spectrum of rHb (αD94A) in the deoxy formshows that the resonance at ˜11.1 ppm (assigned to β37Trp in Hb A) ismissing (FIG. 13C). However, it is not clear what is the new chemicalshift for β37Trp in rHb (αD94A) in the deoxy form. The spectrum of rHb(αD94A, βL105W) in the deoxy form shows an extra proton resonanceappearing at 11.1 ppm compared to that of rHb (αD94A). It appears thatthis resonance originates from β105Trp of rHb (αD94A, βL105W). Thechemical shift for the NH resonance of β105Trp in rHb (βL105W) shiftsupfield 1.7 ppm and closer to the water resonance when α94Asp isreplaced by Ala in rHb (αD94A, βL105W) (FIG. 13C). These resultsindicate that a new H-bond forms between β105Trp and α94Asp in rHb(βL105W) in the deoxy form.

Heteronuclear 2D NMR Studies on ¹⁵N-labeled rHb (βL105W) in the DeoxyForm

In order to confirm the assignment of resonance at 12.7 ppm to β105Trpin the ¹H-NMR spectrum of rHb (βL105W) in the deoxy form, heteronuclear2D NMR experiments on ¹⁵N-labeled rHb (βL105W) in the deoxy form wereperformed (FIGS. is 14 and 15A-15D). FIG. 14 shows the 600-MHz HMQCspectrum of ¹⁵N-labeled rHb (βL105W) in the deoxy form. The ¹⁵N chemicalshift for the proton resonance at 12.7 ppm in the ¹H-NMR spectrum of rHb(βL105W) is at 134 ppm, suggesting that this resonance originates from atryptophan residue. Also observed are the ¹Hδ₁ cross-peaks of Trp ¹⁵Nε₁at (7.8, 134), (7.6, 135), (7.1, 129), and (7.0, 127) ppm for β105Trp,β37Trp, α14Trp and β15Trp, respectively, in the HMQC spectrum of¹⁵N-labeled r Hb (βL105W) in the deoxy form. Also observed are the ¹Hε₁and ¹Hζ₂ cross-peaks of His ¹⁵Nε₂ for α3His at (8.3, 163) and (7.1, 163)ppm, respectively, and for α22His at (7.6, 167) and (7.0, 167) ppm,respectively. The NOESY-HMQC experiments were also performed atdifferent mixing times to provide more evidence for the presentassignments and to investigate the micro-environment for β105Trp in rHb(βL105W) in the deoxy form. For β105Trp (at 12.7 ppm), its ¹Hδ₁ and 1Hζ₂cross-peaks at 7.8 and 8.2 ppm, respectively, can be observed at allfour mixing times (FIGS. 15A-15D). For β37Trp (at 11.2 ppm), its ¹Hδ₁and ¹Hζ₂ cross-peaks at 7.6 and 7.3 ppm, respectively, also can beobserved at all four mixing times (FIGS. 15A-15D). In addition, alsoobserved is the NOE effect between residues of β105Trp and β37Trp in theNOESY-HMQC spectra of ¹⁵N-labeled rHb (βL105 W) in the deoxy form asshown in FIGS. 15A-15D.

The Effects of IHP and Temperature on the Spectra of Hbs in the CO Form

FIGS. 16A and 16B show the exchangeable protons resonances of Hb A, rHb(βL105W), rHb (αD94A, βL105W), and rHb (αD94A) in the CO form in theabsence (FIG. 16A) and presence (FIG. 16B) of IHP at 11, 20, and 29° C.In the absence of IHP, the T marker can be observed only in the spectraof rHb (αD94A, βL105 W) at the lower temperature. In the presence ofIHP, the T marker can be observed in the spectra of all three mutantrHbs. These results have shown that these mutant rHbs can switch fromthe R-structure to the T-structure without changing their ligation statewhen the temperature is lowered and/or when IHP is is added. Besides theappearance of the T marker, the spectra of rHb (βL105W) in the CO formin the presence of IHP also show several differences compared to thosein the absence of IHP. In the presence of IHP, new peaks at 13.1 and11.2 ppm appear to gradually grow from the nearby resonances at 12.9 and11.0 (or 10.8) ppm, respectively, when the temperature is lowered (FIG.16B).

In order to monitor these changes, more detailed HSQC experiments havebeen performed for ¹⁵N-labeled rHb (βL105W) in the absence and thepresence of IHP. In the presence of IHP, the HSQC experiments have alsobeen performed at lower temperatures (FIGS. 17A-17D). In the presence ofIHP at 29° C., the (¹Hε₁, ¹⁵Nε₁) cross-peak at (11.0, 134) ppm. forβ37Trp disappears. The (¹Hε₁, ¹⁵Nε₁) cross-peak at (10.8, 129) ppm forβ105Trp is much weaker in the presence of IHP at 29° C. compared to thatin the absence of IHP (FIGS. 17A and 17B). When the temperature islowered in the presence of IHP, the cross-peak at (11.0, 134) ppmreappears and two new cross-peaks appear at (11.0, 131) and (10.9, 130)ppm (FIGS. 17C and 17D). It appears that these two new cross peaksoriginate from β37Trp and β105Trp.

FIGS. 18A and 18B show the ring-current-shifted proton resonances of HbA, rHb (βL105w), rHb (αD94A, βL105W), and rHb (αD94A) in the CO form inthe absence and presence of IHP at 11, 20, and 29° C. Thering-current-shifted proton resonances of rHb (βL105W) in the CO formdiffer only slightly from those of Hb A in the absence of IHP, whilethey are very different from those of Hb A in the presence of IHP. Thering-current-shifted proton resonances of rHb (αD94A, βL105W) and rHb(αD94A) in the CO form are very different from those of Hb A in both theabsence and presence of IHP, but they are very similar to each other inthe presence of IHP. In addition, the ring-current-shifted protonresonances of rHb (βL105W) in the CO form in the presence of IHP turnout to be very similar to those of rHb (αD94A/ βL105W) and rHb (αD94A)when the temperature is lowered (FIG. 18B). It is believed that thespectra for the ring-current-shifted proton resonances of rHb (αD94A,βL105W) and rHb (αD94A) in the CO form in the presence IHP represent onetype of spectrum for rHbs in the CO form with a stable T-structure.Therefore, the differences in heme pocket conformations between mutantrHbs and Hb A also suggest that these mutant rHbs are much easier toswitch from the R-structure to T-structure in light of the T-marker fromthe exchangeable proton resonances. The resonances at −1.8 and −1.9 ppmhave been assigned to the heme pocket distal valine (E11) of α- andβ-chains of Hb A, respectively (Dalvit, C., et al., Biochemistry 24:3398 (1985) and Craescu, C. T., et al., Eur. J. Biochem 181: 87 (1989),the disclosures of which are incorporated herein by reference). Comparedto the spectra of Hb A, the resonance of distal valine (E11) of β-chainseems to be affected more in the spectra of mutant rHbs, especially inthe presence of IHP, than that of α-chain (FIGS. 18A and 18B). Theseresults imply that the structural switching from the R- to theT-structure induced by IHP, temperature, or the mutations describedherein might occur mainly in the β-chain.

Appropriately cross-linked rHb (βN108Q) and/or rHb (βL105W) can beincorporated into a hemoglobin-based blood substitute or therapeutichemoglobin that is physiologically acceptable for use in clinical orveterinary medicine according to methods know in the art. See, forexample R. M. Winslow, et al. Eds. Blood Substitutes Physiological Basisof Efficacy pp. 82-84 (Birkhauser, Boston, Mass.) (1995), the disclosureof which is incorporated herein by reference. The hemoglobin of thepresent invention may also be advantageously used as a treatment forconditions such as septic shock, prevention of anaphylactic shock duringdialysis.

Although the invention has been described in detail for the purposes ofillustration, it is to be understood that such detail is solely for thatpurpose and that variations can be made therein by those skilled in theart without departing from the spirit and scope of the invention exceptas it may be limited by the claims.

8 1 28 DNA Artificial Sequence DESCRIPTION OF ARTIFICIAL SEQUENCE Primerto introduce betaN108 Q mutation into plasmid pHE2 1 cgtctgctgggtcaggtact agtttgcg 28 2 30 DNA Artificial Sequence DESCRIPTION OFARTIFICIAL SEQUENCE Primer to introduce mutation alphaD94A into plasmidpHE2 2 ctgcgtgttg ctccggtcaa cttcaaactg 30 3 29 DNA Artificial SequenceDESCRIPTION OF ARTIFICIAL SEQUENCE Primer to introduce betaL105 Wmutation into plasmid pHE2 3 ggaaaacttc cgatggctgg gtaacgtac 29 4 27 DNAArtificial Sequence DESCRIPTION OF ARTIFICIAL SEQUENCE Primer tointroduce betaN108 Q mutation into plasmid pHE7 4 acagaccagt acttgtcccaggagcct 27 5 1140 DNA Homo sapiens 5 aaatgagctg ttgacaatta atcatcggctcgtataatgt gtggaattgt gagcggataa 60 caatttcaca caggaaacag aattcgagctcggtacccgg gctacatgga gattaactca 120 atctagaggg tattaataat gtatcgcttaaataaggagg aataacatat ggtgctgtct 180 cctgccgaca agaccaacgt caaggccgcctggggtaagg tcggcgcgca cgctggcgag 240 tatggtgcgg aggccctgga gaggatgttcctgtccttcc ccaccaccaa gacctacttc 300 ccgcacttcg atctgagcca cggctctgcccaggttaagg gccacggcaa gaaggtggcc 360 gacgcgctga ccaacgccgt ggcgcacgtggacgacatgc ccaacgcgct gtccgccctg 420 agcgacctgc acgcgcacaa gcttcgggtggacccggtca acttcaagct cctaagccac 480 tgcctgctgg tgaccctggc cgcccacctccccgccgagt tcacccctgc ggtgcacgcc 540 tccctggaca agttcctggc ttctgtgagcaccgtgctga cctccaaata ccgttaaact 600 agagggtatt aataatgtat cgcttaaataaggaggaata acatatggtg cacctgactc 660 ctgaggagaa gtctgccgtt actgccctgtggggcaaggt gaacgtggat gaagttggtg 720 gtgaggccct gggcaggctg ctggtggtctacccttggac ccagaggttc tttgagtcct 780 ttggggatct gtccactcct gatgctgttatgggcaaccc taaggtgaag gctcatggca 840 agaaagtgct cggtgccttt agtgatggcctggctcacct ggacaacctc aagggcacct 900 ttgccacact gagtgagctg cactgtgacaagctgcacgt ggatcctgag aacttcaggc 960 tcctgggaca agtactggtc tgtgtgctggcccatcactt tggcaaagaa ttcaccccac 1020 cagtgcaggc tgcctatcag aaagtggtggctggtgtggc taatgccctg gcccacaagt 1080 atcactaagc atgcatctgt tttggcggatgagagaagat tttcagcctg atacagatta 1140 6 36 DNA Artificial SequenceDESCRIPTION OF ARTIFICIAL SEQUENCE Primer to introduce betaL105 Wmutation into plasmid pHE7 6 cctgagaact tcaggtggct aggcaacgtg ctggtc 367 1140 DNA Homo sapiens 7 aaatgagctg ttgacaatta atcatcggct cgtataatgtgtggaattgt gagcggataa 60 caatttcaca caggaaacag aattcgagct cggtacccgggctacatgga gattaactca 120 atctagaggg tattaataat gtatcgctta aataaggaggaataacatat ggtgctgtct 180 cctgccgaca agaccaacgt caaggccgcc tggggtaaggtcggcgcgca cgctggcgag 240 tatggtgcgg aggccctgga gaggatgttc ctgtccttccccaccaccaa gacctacttc 300 ccgcacttcg atctgagcca cggctctgcc caggttaagggccacggcaa gaaggtggcc 360 gacgcgctga ccaacgccgt ggcgcacgtg gacgacatgcccaacgcgct gtccgccctg 420 agcgacctgc acgcgcacaa gcttcgggtg gacccggtcaacttcaagct cctaagccac 480 tgcctgctgg tgaccctggc cgcccacctc cccgccgagttcacccctgc ggtgcacgcc 540 tccctggaca agttcctggc ttctgtgagc accgtgctgacctccaaata ccgttaaact 600 agagggtatt aataatgtat cgcttaaata aggaggaataacatatggtg cacctgactc 660 ctgaggagaa gtctgccgtt actgccctgt ggggcaaggtgaacgtggat gaagttggtg 720 gtgaggccct gggcaggctg ctggtggtct acccttggacccagaggttc tttgagtcct 780 ttggggatct gtccactcct gatgctgtta tgggcaaccctaaggtgaag gctcatggca 840 agaaagtgct cggtgccttt agtgatggcc tggctcacctggacaacctc aagggcacct 900 ttgccacact gagtgagctg cactgtgaca agctgcacgtggatcctgag aacttcaggt 960 ggctaggcaa cgtgctggtc tgtgtgctgg cccatcactttggcaaagaa ttcaccccac 1020 cagtgcaggc tgcctatcag aaagtggtgg ctggtgtggctaatgccctg gcccacaagt 1080 atcactaagc atgcatctgt tttggcggat gagagaagattttcagcctg atacagatta 1140 8 146 PRT Homo sapiens 8 Val His Leu Thr ProGlu Glu Lys Ser Ala Trp Thr Ala Leu Trp Gly 1 5 10 15 Lys Val Asn ValAsp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu Leu 20 25 30 Val Val Tyr ProTrp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp Leu 35 40 45 Ser Thr Pro AspAla Val Met Gly Asn Pro Lys Val Lys Ala His Gly 50 55 60 Lys Lys Val LeuGly Ala Phe Ser Asp Gly Leu Ala His Leu Asp Asn 65 70 75 80 Leu Lys GlyThr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys Leu 85 90 95 His Val AspPro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val Cys 100 105 110 Val LeuAla His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln Ala 115 120 125 AlaTyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His Lys 130 135 140Tyr His 145

We claim:
 1. A non-naturally occurring mutant human hemoglobin wherein the asparagine residue at position 108 of the β-chains (SEQ ID NO: 8) is replaced by a glutamine residue.
 2. The hemoglobin of claim 1 possessing low oxygen affinity as compared normal human adult hemoglobin.
 3. The hemoglobin of claim 2 further possessing high cooperativity in oxygen binding comparable to normal human adult hemoglobin.
 4. The hemoglobin of claim 3 further possessing increased stability against autoxidation.
 5. The hemoglobin of claim 1 which is produced recombinantly.
 6. A non-naturally occurring low oxygen affinity mutant hemoglobin with increased stability against autoxidation that has oxygen binding properties comparable to those of human normal adult hemoglobin in the presence of the allosteric effector 2,3-bisphosphoglycerate, wherein the asparagine residue at position 108 of each of the β-chains is replaced by a glutamine residue (SEQ ID NO: 8).
 7. A non-naturally occurring mutant human hemoglobin wherein the asparagine residue at position 108 of the β-chains is replaced by a glutamine residue (SEQ ID NO: 8) wherein said hemoglobin possesses oxygen-binding properties of oxygen affinity as measured by P₅₀ and cooperativity as measured by the Hill coefficient (n_(max)) and similar to those of Hb A in the presence of the allosteric effector 2,3-bisphosphoglycerate as follows: P₅₀ about 17.4 mm Hg, n_(max) about 3.1 in 0.1 M sodium phosphate at pH 7.4 and 29° C.
 8. An artificial mutant hemoglobin which in a cell-free enviornment has oxygen binding properties comparable to those of human normal adult hemoglobin in red blood cells wherein said hemoglobin contains a mutation such that the asparagine residue at position 108 of the β-chains is glutamine (SEQ ID NO: 8).
 9. The hemoglobin of claim 8 which is produced recombinantly.
 10. rHb (βN108Q) (SEQ ID NO: 8).
 11. rHb (βN108Q) (SEQ ID NO: 8) derived from cells transformed with pHE7009.
 12. A non-toxic pharmaceutical composition comprising a non-naturally occurring mutant hemoglobin wherein the asparagine residue at position 108 of the β-chains is replaced by a glutamine residue (SEQ ID NO: 8) in a pharmaceutically acceptable carrier.
 13. The composition of claim 12 wherein said hemoglobin in a cell-free environment has oxygen binding properties lower than those of human normal adult hemoglobin.
 14. The composition of claim 13 wherein said hemoglobin is rHb (βN108Q)(SEQ ID NO: 5).
 15. A method of treating a human subject, comprising administering to said subject a nontoxic composition comprising an artificial mutant hemoglobin, wherein said artificial mutant hemoglobin is rHb (βN108Q) (SEQ ID NO: 8). 